Test for amino-benzene compounds in body fluids



Patented July 16, 1940.

UNITED STATES PATENT OFFICE TEST FOR AMINO-BENZENE- COMPOUNDS IN BODYFLUIDS No Drawing. Application August 10, 1939, Serial No. 289,397

8 Claims.

It is the object of my invention to provide a simple, rapid, andconvenient test for the presence in blood or in urine of amino-benzenechemotherapeutic compounds which contain the sulfanilyl group, which hasa free amino group attached to a benzene ring; and one that can readilybe made by a physician in a sick room without elaborate equipment,without heat, and without the need of other liquid than the blood or theurine itself and conveniently some water, which may be ordinary tapwater.

These amino-benzene chemotherapeutic com pounds are sulfanilamide andsulfapyridine, both of which contain the sulphanilyl group, and the manyderivatives thereof which have been produced and which have varyingdegrees of effectiveness to combat infections. Both sulfanilamide andsulfapyridine contain a free amino group attached to a benzene ring, asthat is inherent in the sulfanilyl group, and so do many if not all oftheir therapeutically efiective derivatives.

These amino-benzene chemotherapeutic compounds, such as sulfanilamideand suliapyridine, are recognized as tremendously important therapeuticagents. But it is essential for their efi'ectiveness that theirconcentration in the blood of the patient be within certain limits. Iftheir concentration in the blood is too low,they are without appreciableeffect in combating the infection; while if such concentration is toohigh, they are also less efiective in combating the disease, and inaddition they then produce certain dangerous effects, both immediatelyand subser quently, such as acidosis, cyanosis, sulfhemoglobinemia,methemoglobinemia, fever, agranulocytosis, and anemia. Thus theseamino-benzene chemotherapeutic agents have both a thresholdblood-concentration and what may be called a safety-ceilingblood-concentration, and it is extremely important that theconcentration of the amino-benzene chemotherapeutic agent in the bloodbe kept between those limits.

In addition, the determination of the concentration of theseamino-benzene chemotherapeutic agents in the blood must be made promptlyif it is to be of any value; for that concentration varies withconsiderable rapidity in a patient. Thus if the test were one which hadto be sent away to a laboratory, for a report in a day or two, it wouldbe practically useless. It is thus essential that the test be one whichcan be made by the physican himself in the sick room, or in thephysicians ofice, in order that he may determine at once his course ofaction.

With sulfanilamide, for instance, it is the present consensus that thethreshold dose is about '7 mg. per 100 cc. of blood, and that thesafetyceiling dose is about 12 mg. per 100 cc. of blood.

If the concentration in the blood is less than about 7 mg. there isordinarily no observable benefit; and if that concentration is aboveabout 12 mg. the eiTect is markedly diminished and dangerous symptomsappear; while if the concentration in the blood is permitted to rise ashigh as 18 or 20 mg. there is almost always cyanosis, and even gravedanger of death.

With sulfapyridine there is as yet considerable difference of opinion asto what the optimum range is; but there is absolute agreement amongphysicians that a concentration much in excess of the safety-ceilingconcentration is dangerous. Probably the optimum concentration range forsulfapyridine has its threshold value somewhere between 4 and 10 mg. andits safety-ceiling I value about 12 to 15 mg. with danger resulting itthe concentration reaches mg.

The situation is further complicated by the fact that the kidneys of thepatient have some tendency and some ability to eliminate theseamino-benzene chemotherapeutic agents; but this tendency and thisability vary markedly in different patients, and in addition many of thediseases being treated tend to impair the action of the kidneys to agreater or less extent. addition, the rate of absorption of theseaminobenzene chemotherapeutic agents from the intestinal tract variesconsiderably with different patients, so that it is not always (if ever)possible to predetermine accurately the concentration of theamino-benzene chemotherapeutic agent in the blood by the size of thedose given.

These are some of the reasons why it is highly desirable that there be asimple, rapid, and convenient test for these amino-benzenechemotherapeutic agents in the blood, and to a lesser extent fordetermining their presence in the urine.

My test can be applied to either blood or urine; and can be made readilyby unskilled persons,

with only a few drops of blood such as may be readily obtained from thepatients finger or ear lobe incidentally to other blood tests, as forredcorpuscle count or for hemoglobin determination, or with only a fewdrops of urine.

that may be available, such as tap water. Because the test is ordinarilymore important as a blood test, rather than as a urine test, I shalldescribe it in the testing of blood.

In either A case any kind of water may be used for dilution In carryingout my invention, I proceed generally as follows:

I dilute approximately two drops of the patients blood about 30 or 40times with any available water, such as tap water. I precipitate theproteins present in this diluted blood; and, desirably but notnecessarily in the same operation, diazotize the amino-benzene compound(such as sulfanilamide or sulfapyridine) present in the blood. For boththe precipitation of the proteins and the diazotization, the dilutedblood is acidulated to a pH at least as low as pH 4.5.

The acidulating agent is most conveniently glutamic acid hydrochloride,or (less desirably) oxalic acid, because they will give the desiredacidity and may be carried in solid form; but, save for that convenienceof availability in solid form, any sufiiciently strong acid can be used,such as hydrochloric acid or sulfuric acid or acetic acid. Theprotein-precipitating agent is most conveniently sodiumhexametaphosphate, which is available on the market under the trademarkCalgon, because it is readily available and may be used in dry form; butin cases where that dry form is not deemed necessary, otherprotein-precipitating agents may be used, such for instance astrichloracetic acid, or sulfosalicylic acid, or a mixture of zincsulfate and sodium hydroxide.

The diazotizing agent is most conveniently sodium nitrite; but othernitrites, such as potassium nitrite or ammonium nitrite, or even nitrousacid, may be used. To facilitate the precipitation of the proteins Ideem it desirable to have also present a coagulating agent, such astalc.

All of this is done most conveniently by using the agents which arementioned above as preferred, and putting them in a single capsule;which may be called Reagent A, and which contains the following:

Sodium nitrite 2 Sodium heXameta-phosphate Glutamic acid hydrochloride30 Talc With such a capsule (Reagent A), the desired proteinprecipitation and the diazotization may be obtained quickly, usually inabout one minute, by simply dumping the contents of one capsule into thediluted blood above described.

The precipitated proteins are removed by filtration; and the clearfiltrate containing the diazotized amino-benzene compound is thenfurther treated, as follows:

The amount of sodium nitrite, or other diazotizing agent used, is anexcess amount, so that there will certainly be enough of it present todiazotize all of the amino-benzene compound which the blood may contain.In consequence, there is some excess nitrous acid in the filtrate. Thisnitrous acid is removed by the addition of a sufficient quantity ofammonium sulfamate; of which the amount is not critical, as an excessdoes no harm. I find it convenient to add the ammonium sulfamate, whichmay be called Reagent B, in the form of a tablet containing 120 mg. ofit. The ammonium sulfamate breaks down the nitrous acid into nitrogenand water, and the nitrogen escapes.

Then I add a definite amount of l-amino-8- naphthol-3,6-disulfonicacidwhich is the socalled H acid of the dye industry. A very smallquantity of this is all that is required, and I actually use 1.5 mg.Because this is such a small amount, I preferably put it in a capsulewith some diluting solids with which it does not react, and which needserve no other purpose although I prefer to include among them somewhich do play a part in facilitating the observation demanded by thetest. This capsule may be called Reagent C, and contains the following:

Mg. 1-amin.o-8-naphthol-3,6-disulfonic acid 1.5 Sodium sulfate 200 Milksugar 20 Talc 30 It is simply necessary to dump the contents of thiscapsule into the clear filtrate after the addi tion of the ammoniumsulfamate tablet.

On this dumping,-with the filtrate acid in character, very littlenoticeable change occurs, although there may be a slight pinkcoloration. This pink coloration is not the test coloration.

The sodium sulfate in the capsule of Reagent C is a mere diluent; butthe talc is to furnish a background for the color that is to be producedat the next step in the test, so that that color will be observablewholly by reflected light and not by transmitted light; and the milksugar serves as a protective colloid to hold the tale in suspension.

Up to this point, the filtrate has been acid in character. At this pointI make it alkaline, to a pH of. between 8.5 and 9.5, by adding somesuitable alkalinizing agent; which is preferably sodium (or potassium)carbonate monohydrate, because it is stable in solid form and isnon-corrosive and non-deliquescent. To get the desired pH, with theproportions given above, I find that a tablet containing mg. of sodiumcarbonate monohydrate is suitable. I may put nothing else in thattablet, but I prefer also to have a little milk sugar co-present, tomake the tablet rapidly soluble; so that I preferably include in each ofthese tablets 60 mg. of milk sugar in addition to the 180 mg. of sodiumcarbonate monohydrate. This tablet may be called Reagent D.

Upon the addition of the tablet containing sodium carbonate, I shake thetube and contents vigorously. There develops at once, as the result ofthe alkalinization, the color which is dependent upon the amount of theamino-benzene compound that is present in the original blood.

Immediately upon the production of that color, it is compared with thecolors on a standard color chart which is graduated in terms ofmilligrams starting with 0 and going at least as high as 15 mg. Withsuch a color chart, the best results of medication with sulfanilamideare probably obtained when the color produced in the filtrate obtainedfrom the blood sample is between a rather light pink which correspondsto 7.5 mg. and a medium dark purple which corresponds to 12.5 mg. Thecolors of the color chart will vary for different amino-benzenecompounds; for while all of those amino-benzene compounds produce muchthe same general range of colors they vary in the amount of theamino-benzene compound required to produce any given. color, and theyalso may vary in the amount which gives the best results of medication.In addition, different physicians will have somewhat different ideas ofoptimum values for any given compound.

In the foregoing example I have given the procedure fortesting theamount of an aminobenzene compound in a blood sample. The same procedureis used for testing the amount of such an amino-benzene compound in a.urine sample. The reading will show the amount of the unacetylatedamino-benzene compound in the urine sample. The total amount of theamino-benzene compound in the urine sample, acetylated and unacetylated,may if desired be determined, although it usually takes more time thanis allowable. To do that, the urine sample is hydrolyzed before the testprocedure starts, as by making the diluted urine barely acid to litmusand then boiling it for about half an hour, to convert any acetylatedamino-benzene compound to the unacetylated form; and then the test isperformed as outlined above.

A quantitative example of making my test on blood is as follows:

Example 0.15 cc. (approximately two drops) of the patients blood,obtained from his finger, is diluted to 6 00., with tap water;conveniently in a test tube marked at 6 cc. and 4 cc. levels. Then thecontents of one capsule of Reagent A are emptied into the test tube, andthe whole is stirred and then allowed to stand for one minute; whereuponthe contents of the test tube are filtered to remove the protein whichhas been precipitated, and the filtrate is further treated. For thatfurther treatment, I use 4 cc. of the filtrate; and to it add one tabletof Reagent B, which is ammonium sulfamate. Then, after about a minute, Iadd the contents of one capsule of Reagent C and one tablet o-f ReagentD, adding Reagents C and D in either order or simultaneously if desired.I at once shake the tube vigorously, and immediately compare the colorproduced with the colors of the standard color chart.

This test can usually be made in less than 5 minutes; and gives thephysician immediate information as to the content of the aminobenzenecompound in the blood so that he may shape his further treatment inaccordance with the indication which the color gives.

I claim as my invention:

1. The process for testing for a sulfanilyl compound in a body fluid ofthe class consisting of blood and urine, which consists in diluting afixed quantity of the body fluid, adding to the diluted body fluid aprotein-precipitating reagent and a diazotizing' reagent and removingthe protein which is precipitated, neutralizing any excess nitrous acidin the filtrate by adding ammonium sulfamate, then adding to thefiltrate enough of an alkalinizing agent to produce a pH between 8.5 and9.5 and a predetermined amount of 1amino-8-naphthol-3,6-disu1fonic acid;to produce a color which by comparison with a color chart indicates thequantity of the sulfanilyl compound in the body fluid.

2. The process for testing for a sulfanilyl compound in a body fluid asset forth inv claim 1, in which the diazotizing agent is sodium nitrite.

3. The process for testing fora sulfanilyl compound in a body fluid asset forth in claim 1, in which the protein-precipitating reagent issodium hexametaphosphate and an acidifying agent.

l. The process. for testing for a sulfanilyl compound in a body fluid asset forth in claim 1, in which the protein-precipitating reagent issodium hexametaphosphate and glutamic acid hydrochloride.

8. A combined diazotant and protein-precipia tant reaction unit, whichreaction unit contains a nitrite, sodium hexametaphosphate, and asolid-form acid sufficient to produce an acidity as great as pH 4.5.

WILLIAM B. FORTUNE.

